Your heart beats ~70 times per minute, with the ventricles ejecting blood during each beat due to calcium-regulated sliding of actin thin filaments past thick filaments composed of tiny myosin molecular motors. Myosin-binding protein C (MyBP-C) is a 140 kD immunoglobulin protein superfamily member that exists within the myosin thick filament. MyBP-C is a critical modulator of the heart's pumping capacity, which is emphasized by genetic mutations being a leading cause of familial hypertrophic cardiomyopathy and sudden death; most notably in young athletes. Despite its clinical impact, the underlying molecular mechanics by which MyBP-C tunes cardiac contractility in healthy hearts is not well understood. Therefore, understanding such mechanisms under normal conditions is necessary to determine how mutations affect its modulatory capacity. With the support of his mentorship team, Dr. Previs will acquire new technical skills and combine state-of-the-art, single molecule biophysical techniques, in vitro protein expression, and quantitative proteomics to define the molecular basis for MyBP-C's functional impact on calcium-dependent actomyosin interactions using native thick and thin filaments isolated from transgenic mouse and failing human hearts. In an effort to unravel MyBP-C's molecular impact on cardiac contractility, Dr. Previs developed a total internal fluorescence microscopy (TIRFM) assay to visualize single actin filaments sliding over native thick filaments from transgenic mouse hearts, with the guidance of Dr. David Warshaw, an expert in single molecule biophysics. Through a combination of molecular biophysics, mass spectrometry-based proteomics, and analytic modeling, he gathered direct molecular evidence that MyBP-C's N-terminal domains interact with actin and/or the myosin head to slow actin filament sliding only where MyBP-C exists within the thick filament. Thus he demonstrated that MyBP-C acts like a governor in a car engine to limit the heart's pumping power. Both the literature and Dr. Previs' current research (submitted to PNAS) suggests that before applying the molecular brakes, MyBP-C's N-terminal domains rev up the heart during the early stages of contraction by activating calcium-regulated thin filaments at low calcium levels, through an independent molecular mechanism. With additional mentoring from Dr. Warshaw and Drs. Jeffrey Robbins and Kathleen Trybus, having expertise in mouse transgenesis, molecular biology and in vitro protein expression, he is proposing to determine if MyBP-C's activation and inhibition of thin filament sliding involve unique MyBP-C N-terminal domains that specify actin and/or myosin S2 binding. With his mentoring team, he will then develop a novel laser trap-based TIRFM assay to observe the sequence of events by which a single fluorescently-tagged MyBP-C molecule binds to a calcium-regulated thin filament and turns it on so that myosin motors will bind under low calcium conditions, where binding is normally inhibited. This assay will have broad implications for investigating thin filament regulation by muscle biologists and for continued use throughout his independent career. Specifically, during the independent phase of the award (R00) and beyond, he will combine his graduate training in quantitative mass spectrometry with the biophysical and molecular biological tools and knowledge gained during the mentored phase of the award (K99), to define MyBP-C's role in altering the contractility of failing human myocardium. These studies will benefit from his direct access to the world's largest human cardiac tissue bank (Sydney Heart Bank, Australia) run by Dr. Cris dos Remedios, an expert in fluoresce spectroscopy of actin binding proteins and the molecular basis for heart failure. The inclusion of Dr. Remedios on Dr. Previs' Mentoring Committee during the K99 phase will provide guidance for scientific and career development, and their independent collaboration (R00) will provide Dr. Previs with human myocardium for his studies and exposure to an international group of scientists who utilize the tissue bank to address similar scientific questions from differing perspectives. The science generated by the proposed studies will advance our understanding of MyBP-C's molecular mechanics, build consensus between conflicting molecular models within the field (i.e. actin and/or myosin binding), and provide a critical translational link to human heart failure, where additional thick and thin filament compensatory and/or decompensatory regulatory mechanisms are at play. This award will provide Dr. Previs with a means to acquire new technical skills necessary to address both his short- and long-term hypotheses, and mentorship in career development to obtain his long-term goal of becoming a successful independent investigator at a prestigious academic institution.